Anesthetic exposure during synaptogenesis in the developing brain causes widespread neurodegeneration, electrophysiologic abnormalities of neuronal networks and long-term cognitive deficits. To date, delayed cognitive deficits have been attributed to anesthetic induced neuronal apoptosis. Unpublished work from our laboratory has shown that prevention of neuronal apoptosis does not prevent anesthetic-mediated memory deficits, suggesting that factors other than apoptosis, including development of neuronal networks, play a central role in anesthetic induced delayed cognitive dysfunction. Using a well-characterized model of hippocampal mossy fiber development, we have begun preliminary investigations on the effect of anesthetics on network formation. Mossy fibers originate in dentate granule cells, traverse via the supra and infra pyramidal bundles (SPM, IPM) to synapse on to CA3 neurons. Our preliminary data indicate that propofol leads to a dramatic loss of SPM and IPM bundles with a consequent of loss of synapses onto CA3. The mechanism that underlies loss of mossy fibers is not clear. The development of axons requires active axonal growth with appropriate targeting and formation of synapses. The axonal growth cone at the tip of the axon is required for growth and proper pathfinding. Propofol causes a dramatic collapse of the growth cone, thereby preventing axon extension. The growth cone has a dynamically regulated actin cytoskeleton, which is essential for axonal growth. RhoGTPases, which include RhoA, Rac1 and Cdc42, are key regulators of the actin cytoskeleton. Our data indicate that there is an imbalance between RhoA and Rac1-Cdc42 activity following propofol exposure such that actin is depolymerized, thereby leading to growth cone collapse. Importantly, inhibition of RhoA prevents growth cone collapse. Anterograde and retrograde transport of material (e.g., signaling endosomes, mitochondria) is essential for axonal growth and RhoGTPases are also required for transport. Rac1 activation mediated cofilin activation is necessary for axonal transport; by contrast, RhoA activation leads to downstream activation of RhoA kinase (ROCK), LIM kinase (LIMK), cofilin deactivation and inhibition of transport. Propofol, by increasing RhoA and reducing Rac1-Cdc42 activity, significantly inhibits transport. Given that propofol has adverse consequences on the processes (growth cone, guidance, axonal transport) that are critical for axonal growth and neuronal network development via an imbalance in RhoGTPase, it is conceivable that restoration of the balance of RhoA and Cdc42-Rac1 activity might mitigate the advance effects of propofol on network development. Accordingly, the central hypothesis of the present proposal is that during the vulnerable period of PND5-7, anesthetic exposure leads to RhoA activation, Rac and Cdc42 inhibition, disruption of the cytoskeleton, growth cone collapse and impaired axonal transport, all of which leads to aberrant synaptic connections and to long term cognitive dysfunction.